Related Experiment Videos
Modifying specificity of antidigoxin antibodies using insertional mutagenesis
Rustem A Krykbaev1, Panayota Tsantili, Philip D Jeffrey
1Antibody Engineering Laboratory, Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston 02114, USA.
Protein Science : a Publication of the Protein Society
|November 21, 2002
Summary
Antibody engineering created new antibodies that preferentially bind digitoxin over digoxin by altering specific regions. These modified antibodies show significantly enhanced binding to digitoxin and related compounds.
Area of Science:
- Immunology
- Biochemistry
- Structural Biology
Background:
- Antibodies (Abs) against digoxin exhibit varying specificities for digoxin analogs.
- Antibody 26-10 binds equally to digoxin and digitoxin, differing by a C12 hydroxyl group.
- Other antidigoxin Abs can differentiate ligands by orders of magnitude.
Purpose of the Study:
- To engineer antibodies with preferential binding to digitoxin over digoxin.
- To investigate the role of LCDR3 insertions and mutations in antibody specificity.
- To enhance antibody binding to cardiac glycoside analogs.
Main Methods:
- Generated bacteriophage display libraries with randomized LCDR3 and insertions.
- Selected mutant antibodies by panning against digoxin and analogs.
- Utilized site-directed mutagenesis to optimize binding affinity and specificity.
Main Results:
- Engineered mutants exhibited up to 47-fold greater preferential binding to digitoxin compared to digoxin.
- Identified a consensus sequence in high-affinity digitoxin binders, including L:Trp94 substitution.
- Optimized mutants with insertions and L:Trp94 mutation maximized digitoxin binding.
- Selected mutants showed up to 600-fold increased relative binding to gitoxin.
Conclusions:
- LCDR3 insertions and specific mutations can engineer antibody specificity for cardiac glycosides.
- Antibody 26-10 can be modified to create Abs with high affinity for digitoxin and gitoxin.
- Structural insights guided the engineering of enhanced antibody-ligand interactions.